The studies performed here provide important new insights into the mechanism of NOD1 signaling and the role of NOD1 in protection against infection at mucosal surfaces. With respect to signaling we show that NOD1 activated by purified NOD1 ligand (?DGDAP) induces chemokine production via a pathway primarily induced by type I IFN, namely the activation of a heterotrimeric transcription factor complex composed of Stat1, Stat2 and IRF9 known as ISGF3. Furthermore, we show that this pathway is initiated by an interaction between RICK and TRAF3 that leads to activation of the TRAF3 down-stream components, TBK1 and IKKepsilon and, in turn, to the activation of IRF7 and the production of IFN-beta;the latter then activates the aforementioned ISGF3 pathway. With respect to function, we show that NOD1 activation leads to induction of chemokines associated with the Th1 response (IP-10 and I-TAC) as well as IFN-beta and in vivo inhibition of the ISGF3 pathway via administration of Stat1 siRNA greatly impairs host defense against H. pylori infection of the gastric mucosa. In addition, loss of IFN-beta signaling due to IFN-beta receptor deficiency has a similar effect. Thus, these studies establish the somewhat unexpected finding that NOD1 signaling utilizes the type I IFN-induced ISGF3 pathway to enable host defense. Evidence that activation of NOD1 leads to Th1 chemokine production (IP-10 and I-TAC) mainly via a Type 1 interferon signaling pathway was based on both positive and negative findings obtained in studies of both cell lines and primary cells. First, specific inhibitors of ERK, and JNK activation had no effect and an inhibitor of NF-kappaB had only a marginal effect on NOD1-mediated chemokine production, whereas an inhibitor of RICK clearly blocked such production. Second, phosphorylation of p38 MAP kinase or JNK was not observed in cells stimulated with NOD1 ligand, and in studies of nuclear translocation of the NF-kappaB subunits, a sensitive mobility shift analysis was weakly positive and a Transfactor binding assay was negative. In contrast, NOD1 stimulation led to robust activation and translocation of all three components of ISGF3, namely, Stat1, Stat2 and IRF-9. Third, transfection of cells with siRNA specific for RICK, Stat1 or Stat2 led to down-regulation of chemokine production, whereas transfection of siRNA specific for p65 or p38 MAP kinase had little or no effect on such production;in addition, siRNA specific for TBK1 and IKKepsilon also down-regulated chemokine production as did blockade of type I IFN signaling by anti-IFNalpha/betaR. Extensive in vivo studies of NOD1 signaling including studies of such signaling in relation to H.pylori infection provided important in vivo corroboration of the above in vitro studies. Initial studies showed that stimulation of mice with NOD1 ligand induced a robust IFN-? response along with an IP-10 response arising from non-hematopoietic cells. Further studies showed that the increased susceptibility of NOD1-deficient mice to H. pylori infection was associated with a decreased ability to produce IFN-beta and IP-10, both factors dependent on the ISGF3 signaling pathway, and, in fact infected gastric tissue manifested evidence of decreased activation of components of this pathway. In contrast, these mice produced ample amounts of NF-kappaB-dependent chemokines and cytokines and their infected gastric tissue exhibited NF-?B activation equivalent to that in NOD1-intact mice. Thus, while NF-kappaB activation does occur in the gastric mucosa infected with H. pylori, such activation is not dependent on NOD1. This conclusion is in line with our finding that H. pylori also activates NF-kappaB when added to cultures of epithelial cells. As shown by Hirata et al., this NF-kappaB activation by H. pylori is independent of NOD1. These data were then augmented by studies addressing the causal relation between between ISGF3 signaling and H.pylori infection in which it was shown that administration of Stat1-specific siRNA inhibited up-regulation of lP-10 and IFN-beta production in response to H.pylori infection and led to a decreased ability to control such infection on the gastric mucosal surface. By contrast, down-regulation of NF-kappaB in vivo by administration of NF-?B decoy ODN did not inhibit the up-regulation of IP-10 or IFN-? production in response to H.pylori infection;in addition it led to only a minor decrease in the ability of the treated mouse to control such infection that was not necessarily related to effects on NOD1 signaling since the decoy ODN also down-regulated TNF production. Since the NF-?B decoy ODN has previously been shown to suppress the activity of all NF-kappaB components (p50, p65, c-Rel, p52 and RelB), this study also rules out the possibility that NOD1 signals through a non-canonical NF-?B pathway. A key finding in this study was that NOD1 activation results in a form of RICK that binds to TRAF3 and thereby initiates signaling through TBK1/IKKepsilon and IRF7 to induce IFN-beta. The interaction of NOD1-activated RICK with TRAF3 (rather than with TRAF6) results in activation of a TRAF that has been shown to profoundly suppress both the canonical and non-canonical NF-kappaB pathway, thus providing an explanation for why the latter pathway is not utilized in NOD1 activation. It should be noted, however, that our data does not answer the question of why NOD1-activated RICK interacts preferentially with TRAF3 and not with TRAF6, as does NOD2-activated RICK. One mechanism we considered was that NOD1 induces RICK kinase function which then phosphorylates TRAF3;however, we found that this cannot be the case since mutations of RICK leading to loss of all kinase function does not impair the ability of RICK to mediate NOD1 ligand induced IP-10 synthesis in HT-29 cells (unpublished data). This negative finding, however, allowed us to rule out that NOD1-activated RICK directly activates ISGF3 by phosphorylating Stat1 and Stat2 (rather than acting via TRAF3 and IFN-beta). Finally, while NOD1 induction of chemokines via an ISGF3-mediated pathway may seem unusual, the use of this pathway (and not the NF-?B pathway) has previously been demonstrated with respect to LPS signaling. In the latter case, it has been shown that LPS induces only low amounts of IP-10 (and certain other chemokines) in the absence of type I IFN. Presumably, LPS enters the type I IFN/ISGF3 system via TRIF activation of TRAF3. These studies along with the present studies show that ISGF3 transactivation of the IP-10 promoter at an ISRE site is sufficient for robust IP-10 transcription even though the promoter does contain NF-kappaB binding sites. Overall then, the combined in vitro and in vivo data summarized above strongly support the notion that NOD1 induction of chemokines associated with the Th1 response by gastrointestinal epithelial cells depends mainly on the type I IFN/ISGF3-pathway.